Carbazole derivatives

ABSTRACT

Novel tethered hapten intermediates and related conjugates based on carbazole and/or dibenzofuran, as well as methods for making and using such conjugates. Haptens based on the above core structures may be substituted at any position on the aromatic rings with a wide variety of substituents. Using tethered intermediates, immunogens, tracers, solid supports and labeled oligonucleotides are all described; as are methods for using the intermediates to prepare the conjugates, methods of using the conjugates to make and purify antibodies, as assay tracers, and in nucleic acid hybridization assays. Kits containing haptenated oligonucleotides and anti-hapten conjugates are also described.

This application is a division of U.S. patent application Ser. No.08/084,495, filed Jul. 1, 1993, now U.S. Pat. No. 5,464,746, which is acontinuation-in-part of U.S. Ser. No. 07/808,839 filed Dec. 17, 1991,now abandoned the whole of which is incorporated by reference.

The present invention relates to novel carbazole and dibenzofuran haptencompounds, to tethered intermediates, to immunogens useful for preparingantibodies, to tracer compounds useful for assaying the haptens, tooligonucleotides labeled with the haptens and to kits containing thesereagents. The invention also relates to various methods for makingand/or using the novel haptens and the derivatives specified above.

I. BACKGROUND OF THE INVENTION

It is commonly known that many small molecules will not elicit anantibody response by themselves but, when coupled to an appropriateimmunogenicity conferring carrier molecule (to become an immunogen),antibodies can be prepared against the hapten. This technology isdiscussed in many textbooks. See Erlanger, B. F. in Methods ofEnzymology, 70:85-105 (Academic Press 1980); and Hum, B. A. L., et al.,in Methods of Enzymology, 70:105- (Academic Press 1980).

Many methods of adding haptens to oligonucleotide probes are known inthe literature. A review of such conjugate literature is found inGoodchild, Bioconjugate Chemistry, 1(3): 165-187 (1990). EnzoBiochemical (New York) and Clontech (Palo Alto) both have described andcommercialized probe labeling techniques, including techniques forlabeling probes with biotin or similar haptens. In addition, co-pendingapplications U.S. patent application Ser. Nos. 625,566, filed Dec. 11,1990 now abandoned and 630,908, filed Dec. 20, 1990 now U.S. Pat. No.5,290,925 teach methods for labeling probes at their 5" and 3" endsrespectively. The entire disclosures of the aforementioned co-pendingapplications are incorporated by reference. The hapten label or "hook"may be used either to isolate a desired target sequence (i.e. byhybridization with a haptenated oligonucleotide and collection of thehaptens with a specific binding partner); or to attach a detectablesignaling moiety to a target sequence (e.g. by probing target with ahaptenated oligonucleotide and using an anti-hapten conjugate with adetectable signal generating compound such as a fluorophore,chemilumiphore, colloidal particle or enzyme).

According to one known method for labeling an oligonucleotide, alabel-phosphoramidite reagent is prepared and used to add the label tothe oligonucleotide during its synthesis. For example, see Thuong, N. T.et al., Tet. Letters, 29(46):5905-5908 (1988); or Cohen, J. S. et al.,U.S. patent application Ser. No. 07/246,688 now abandoned (NTIS ORDERNo. PAT-APPL-7-246,688) (1989). However, DNA synthesis reactionconditions are quite severe (e.g. iodine oxidation and ammoniumhydroxide cleavage) and many haptens (e.g. biotin and fluorescein) donot readily withstand these conditions without modification. In anotherapproach useful for labile haptens, a linker having a protected terminalamine is attached to the desired end of the oligonucleotide. The aminecan be deprotected and, under milder conditions, reacted with a label.

Automated synthesis of oligonucleotides (See e.g. Beaucage andCaruthers, Tet. Letters, 22(20):1859-1862 (1981) and U.S. Pat. Nos.4,973,679 and 4,458,066) is often the most efficient method of preparingprobes. However, the hostile conditions required during automatedsynthesis limit the choice of labels available for labeling by thismethod. The present invention overcomes these drawbacks by describingnovel haptens which will withstand the rather rigorous conditions of DNAsynthesis. Thus, using the haptens of the invention, an oligonucleotidecan be directly labeled during automated synthesis, without involving anintervening isolation or a secondary labeling reaction.

The invention has a further advantage in that successfully labeledoligonucleotides can easily be isolated from unlabeled oligonucleotidesby an affinity separation method using a specific binding partner, e.g.an antibody, for the hapten.

Methodology for preparing tracer molecules also is known. For example,fluorescence polarization assays require tracers comprising ananalyte-hapten coupled to a fluorescent molecule. Typically, theanalyte-hapten and a known amount of tracer are allowed to compete for alimited amount of a specific binding member for the hapten, and thelabeled tracer is thereby partitioned between a bound and free form. Thesignal from the bound form is differentiable from the signal from thefree form, so that the amount of analyte-hapten can be estimated. Onemethod for differentiating the signals is by fluorescence polarizationimmunoassay (FPIA), in which the "millipolarization", the "span" or the"relative intensity" can be measured as described in the literature andbelow. The technique of FPIA has been described, for example, in Jolley,M. E., J. Analyt. Toxicol., 5:236-240 (1981) and in Blecka, L. J. Amer.Assoc. Clin. Chem. pp. 1-6 (March 1983), the entire disclosures of whichare incorporated herein by reference.

Some researchers have prepared antibodies to (i.e. demonstratedimmunogenicity of) polyhalogenated dibenzofurans in the context of toxicbyproduct detection and cleanup. For example, Vanderlaan, et al haveworked with polyhalogenated p-dioxins (which differ from dibenzofuran inhaving a six-member, 2 oxygen spacer ring between the two benzene rings,instead of a 5 member, one oxygen furan) and polyhalogenateddibenzofurans; see e.g. U.S. Pat. No. 4,798,807, EP-A-332 819,Chemosphere, 16(8-9): 1635-39 (1987), Toxicology, 45(3):229-43 (1987).U.S. Pat. No. 4,238,472 to Albro, et al., describes antibodies to andimmunogens made from similar polychlorinated compounds. Albro, et al.show that antisera to the polychlorinated dioxins and dibenzofurans hadvery little reactivity with the unchlorinated species.

Pandey, et al., J. Immunol. Methods, 94(1-2):237-46 (1986), describeantibodies to and immunogens made from 3-azido-N-ethylcarbazole.

However, applicants are unaware of any an demonstrating the antigenicityor immunogenicity of the claimed carbazole and dibenzofuran derivatives.

II. SUMMARY OF THE PRESENT INVENTION

In one aspect, the present invention is derived from the class ofcompounds which are based carbazole and dibenzofuran derivatives of thefollowing structure: ##STR1## wherein a and a' when taken alone are 1 to4 groups independently selected from the group consisting of: hydrogen,C₁ -C₁₀ -alkyl, C₁ -C₁₀ -alkoxy, C₁ -C₁₀ -alkylthio, halo-C₁ -C₁₀-alkyl, C ₁ -C ₁₀ -alkylamino, di-(C ₁ -C₁₀ -alkyl)amino, aryl C₁ -C₁₀-alkyl, optionally substituted aryl, halogen, amino, carboxy,carboxamido, hydroxy, mercapto, nitro, nitroso, sulfo, phospho andprotected forms thereof; or alternatively a and a' when adjacent andwhen taken together with the carbons to which they are joined form afused ring;

G is selected from S,O and NR wherein R is hydrogen, C₁ -C₁₀ -alkyl,optionally substituted aryl, optionally substituted sulfonyl,thiophenyl, carboxy, carboxamido, and protected forms thereof; and

A is a linking moiety of the formula -L-y, wherein y is a functionalgroup that can react directly or after activation with functional groupsin a second molecule and L is spacer group consisting of from 1 to about50 atoms.

In another aspect, the present invention relates to conjugate compoundshaving the following structure: ##STR2## wherein a and a', G and A areas defined above; and Q is an immunogenicity conferring carriermolecule, a detectable label, an oligonucleotide or a solid support.

In another aspect, the invention relates to antibodies, eitherpolyclonal or monoclonal, which are reactive with the above compounds(I) or (II). Such antibodies may be prepared by the process of injectingan immunogen (II) into an animal and recovering the antibodies.

In addition, the invention relates to the following methods of using theabove compounds:

1. Use of compounds (II) to prepare immunogens, tracers, labeledoligonucleotides and affinity solid supports;

2. Use of compounds (III: Q=immunogenicity conferring carrier) to raiseantibodies;

3. Use of compounds (III: Q=solid support) to isolate or purifyantibodies;

4. Use of compounds (III: Q=oligonucleotide) for detection of nucleicacids complementary to the oligonucleotide; and

5. Use of compounds (III: Q=detectable signal moiety) for detection ofhapten-analog analytes.

Finally, the invention also relates to kits containing compounds of theinvention (e.g. II:y=phosphoramidite or III: Q=oligonucleotide), incombination with an antibody reactive with the compounds, said antibodybeing attached to or adapted for attachment to either solid supports ordetectable labels. In the second example, the oligonucleotide probe maybe hybridized with a target and the antibody may be used to separate ordetect it. In the first example, the phosphoramidite may be used tolabel one's own oligonucleotide during its synthesis, while the antibodyis used as before.

III. DETAILED DESCRIPTION

The following definitions are applicable to the present invention:

"Antigen" is defined in its usual sense, to refer to a molecule orcompound which is capable of eliciting an immune or antibody response ina challenged animal. Compounds which are not antigenic by themselves cansometimes by made to elicit the immune response by coupling the compound(a "hapten") to an "immunogenicity conferring carrier" molecule to forman "immunogen". While such haptens are not "antigenic" in the strictsense, they are capable of imitating antigens and have many propertiesin common with antigens. Thus, the terms antigen and hapten are oftenused interchangeably. For example, both haptens and antigens have atleast one "determinant" which, as used herein, refers to a region of theantigen or hapten which is involved in specific binding reactionsbetween the antigen or hapten and an antibody. Some haptens and antigenshave more than one determinant region or site and thus are "polyvalent".In essence, it is the determinants which differentiate antigens, andtherefore, antibodies from one another on the basis of immunologicalspecificity.

For purposes of this application, "hapten" is defined as any compoundhaving the core structure shown below: ##STR3## wherein a and a' whentaken alone are 1 to 4 groups independently selected from the groupconsisting of: hydrogen, C₁ -C₁₀ -alkyl, C₁ -C₁₀ -alkoxy, C₁ -C₁₀-alkylthio, halo-C₁ -C₁₀ -alkyl, C ₁ -C ₁₀ -alkylamino, di-(C ₁ -C₁₀-alkyl)amino, aryl-C₁ -C₁₀ -alkyl, optionally substituted aryl, halogen,amino, carboxy, carboxamido, hydroxy, mercapto, nitro, nitroso, sulfo,phospho and protected forms thereof; or alternatively a and a' whenadjacent and when taken together with the carbons to which they arejoined form a fused ring;

G is selected from S, O, and NR wherein R is hydrogen, C₁ -C₁₀ -alkyl,optionally substituted aryl, optionally substituted sulfonyl,thiophenyl, carboxy, carboxamido, and protected forms thereof.

As suggested above, the term "immunogen" refers to a conjugate of ahapten or antigen and a carrier molecule. The carrier is often a proteinor peptide. Known immunogenicity conferring carriers include, forexample, naturally occurring poly(amino-acids), albumins and serumproteins such as bovine thyroglobulin (BTG), globulins, lipoproteins,ocular lens proteins, bovine serum albumin (BSA), keyhole limpethemocyanin (KLH), egg ovalbumin, bovine gamma globulin (BGG), thyroxinbinding globulin (TBG), and the like. Alternatively, syntheticpoly(amino-acids) can be utilized such as polylysine, etc. However, anymolecule which is capable of conferring antigenicity to a hapten is an"immunogenicity conferring carrier."

The term "hapten-specific binding member", as used herein, refers to amember, such as an antibody or receptor, that specifically binds to thehapten. The determinants on the hapten are responsible for the specificbinding of the binding member to the hapten. The most common and usualspecific binding member is an antibody, either polyclonal or monoclonal.

In general, terms like "alkyl", "alkenyl" and "aryl" have the meaningsusually attributed to them by persons skilled in the art of organicchemistry. For example, alkyl refers to monovalent straight or branchedaliphatic radicals which may be derived from alkanes by the removal ofone hydrogen, and have the general formula C_(n) H_(2n+1). Alkylsubstituents may have from 1 to about 30 carbons, more practically 1 toabout 20. "Lower alkyl" refers to alkyls having from 1 to about 10carbons. Examples of lower alkyl include CH₃ --, CH₃ CH₂ --, CH₃CH(CH₃)--, and CH₃ (CH₂)₄ --.

"Alkylene" refers to a divalent group derived from a straight orbranched chain saturated hydrocarbon by removal of two hydrogen atoms.Examples include methylene, 1,2,-ethylene, 1,3-propylene,2,2-dimethylpropylene and the like.

"Halo-C₁ -C₁₀ -alkyl" refers to a lower alkyl group, as defined below,bearing at least one halogen substituent, for example chloromethyl,fluoromethyl, chloroethyl, trifluoromethyl and the like.

"C₁ -C₁₀ -alkoxy" refers to an alkyl group, as defined above, which isbonded through an oxygen atom. Examples of such alkoxy groups includemethoxy, ethoxy, t-butoxy and the like.

"C₁ -C₁₀ -alkylamino" refers to amino groups substituted with one or twolower alkyl groups, as defined above, including methylamino, ethylamino,dimethylamino, diethylamino, propylamino and ethylmethylamino.

"C₁ -C₁₀ -alkylthio" refers to thio groups substituted with one or twolower alkyl groups, as defined above, including methylthio, ethylthio,dimethylthio, and diethylthio.

"Alkenyl" refers to monovalent straight or branched aliphatic radicalswhich may be derived from alkenes by the removal of one hydrogen, andhave the general formula C_(n) H_(2n-1). Alkenyl substituents may havefrom 1 to about 30 carbons, more practically 1 to about 20. "Loweralkenyl" refers to alkenyls having from 1 to about 10 carbons."Olefinic" is a synonym for alkenyl.

"Aryl" refers to a monovalent radical derived from aromatic hydrocarbonsor heteroaromatic compounds by the removal of one hydrogen. Arylsubstituents have ring structures, such as those of phenyl, naphthyl and2-thienyl. Typically, aryl substituents are planar with the π electronclouds of each carbon remaining on opposite sides of the plane. Arylsubstituents satisfy the Huckel (4n+2) π electrons rule.

"Arylalkyl" refers to an aryl group as defined above substitute with oneto two lower alkyl groups as defined above.

Protecting groups are defined as groups that can be removed underspecific conditions, but which shelter or hide a reactive atom orfunctionality temporarily during intermediate reactions under otherconditions. Protecting groups for hydroxyl, amino and thiolfunctionalities are well known in the art (T. W. Greene, ProtectiveGroups in Organic Synthesis, John Wiley and Sons, NY, 1981). Hydroxylfunctions are routinely protected as alkyl or aryl ethers alkyl, aryl,alkenyl), silyl ethers (silyl), esters (acyl), carbonates(C(═O)--O--alkyl, --C(═O)--O--aryl, --C(═O)--O--alkenyl) and carbamates(C(═O)--NH-alkyl, --C(═O)--NH-aryl, --C(═O)--NH-alkenyl). Aminofunctions are routinely protected as carbamates (--C(═O)--O--alkyl,--C(═O)--O--aryl,(C(═O)--O--alkenyl), amides (C(═O)--alkyl,--C(═O)--aryl, --C(═O)--alkenyl), cyclic imides (phthaloyl), N-benzylderivatives (--CH.sub.(n) aryl.sub.(3-n), n=1-3), imine derivatives(═CH.sub.(n) alkyl.sub.(2-n), ═CH.sub.(n) aryl.sub.(2-n) n=0-2), silylderivatives (silyl), N-sulfenyl derivatives (S-aryl, --S--CH.sub.(n)aryl.sub.(3-n), n=0-3), and N-sulfonyl derivatives (--SO₂ -aryl, --SO₂-alkyl). Thiol functions are routinely protected as thioethers(--CH.sub.(n) aryl.sub.(3-n), n=1-3, alkyl), thioesters (acyl),thiocarbonates (C(═O)--O--alkyl, --C(═O)--O--aryl, --C(═O)--O--alkenyl),thiocarbamates (C(═O)--NH-alkyl, --C(═O)--NH-aryl, --C(═O)--NH-alkenyl),and disulfides (--S--alkyl, aryl). Where more than one protecting groupis called for, it will be understood that each group may beindependently selected from the various protecting groups. Indeed, oneof ordinary skill in the art will know which protecting groups areroutine for which functional groups.

As used herein, the "linking moiety" A is also referred to as a"tether". These terms refer to the spacer molecule having the formula:

    --L--y

where y is a reactive functional group that can react directly or afteractivation with the functional groups in a second molecule, e.g. theconjugation partner, Q; and L is a spacer group consisting of from 1 to50 carbon and heteroatoms. Typically, L will include not more than tenheteroatoms, and will be arranged in a straight or branched chain orcyclic moiety, saturated or unsaturated, with the provisos that not morethan two heteroatoms may be directly linked in the sequence --L--y, thatthe sequence --L--y cannot contain --O--O-- linkages, that cyclicmoieties contain 6 or fewer members, and that branchings may occur onlyon carbon atoms. In formulas where A is already linked to Q, the group yis dropped from the formula, leaving A =--L--.

Typically, y is chosen from the group consisting of hydroxy (--OH),thiol (--SH), carboxy (--C(═O)OH), amino (--NH₂), aldehyde (--CH(═O)),leaving group, Michael acceptor, phosphoramidite, phosphonate andprotected forms of these functional groups. In synthesis, the linkingmoiety often comprises a bifunctional compound designated x--L--ywherein x is also a functional group (selected from the same group as y)which can react with functional groups on the hapten or --R. Manybifunctional linkers are known to one skilled in this art. For example,heterobifunctional linkers are described in, e.g. U.S. Pat. No.5,002,883 (Bieniarz). These are preferred in some cases due to thespecificity of their ends for one functional group or another.

A "Michael acceptor" is defined in the art as follows: "The nucleophilicaddition of enolate (or analogous) anions to the carbon-carbon doublebond of α,β-unsaturated ketones, aldehydes nitriles or carboxylic acidderivatives, [is] a process known as the Michael reaction . . . Theunsaturated compounds in the reaction, often called Michael acceptors,may include any unsaturated system having a functional group capable ofstabilizing the carbanionic intermediate . . . The Michael acceptors mayalso add a variety of nucleophiles such as alcohols, thios, and amines."H. O. House, Modern Synthetic Reactions, W. A. Benjamin, Inc., MenloPark Calif, 1972, pp. 595-96. Common functional groups which canactivate a double bond to this kind of nucleophilic addition (therebyforming Michael acceptors) include, --CH(═O), --C(═O)R†, --C(═O)NH₂,--CN, --NO₂, --S(═O)R†, --S(═O)₂ R†, wherein R†, can be alkyl or aryl.Thus, exemplary Michael acceptors include: ##STR4## wherein d, e, and fcan independently be hydrogen, alkyl, or aryl, and wherein U is chosenfrom --CH(═O), --C(═O)R†, --C(═O)NH₂, --CN, --NO₂, --S(═O)R†, and--S(═O)₂ R†, and R†is again alkyl or aryl. A particularly preferredMichael acceptor is maleimide: ##STR5##

Solid supports refer to a wide variety of support materials. Polymericplastics, such as polystyrene, polypropylene, andpolytetrafluoroethylene, are exemplary. Glass is also a useful support.Supports may take any size or shape, including beads, microparticles,tubes, rods, plates, wells and cuvettes. Supports may include functionalgroups for conjugation, or may be derivatized prior to conjugation.Alternatively, supports may be coated or adsorbed in some cases.Supports should be physically separable from reagent solutions, based onsize, weight, shape, charge, magnetic properties or some other physicalproperty. It will be realized that two distinct uses for solid supportsare described herein. First, antibodies can be purified using supportsconjugated to tethered intermediates; and secondly, supports to whichanti-hapten antibodies are attached are useful for separating and/ordetecting oligonucleotides labeled with haptens, and for competitivehapten-analog assays.

Antibodies are prepared by developing an immune response in animals tothe immunogens described hereinafter. The immunogen is administered toanimals such as rabbits, mice, rats, sheep or cows by a series ofinjections according to techniques generally known in the art. Anantibody, according to the present invention, is raised in response toan immunogen of the invention which is derived from the haptensdescribed above. Both polyclonal and monoclonal antibodies recognizespecific epitopes on an immunogen, and, while typically polyclonalantibodies have been utilized in the present invention, both may besuitable. Polyclonal antibodies consist of a mixture of multipleantibodies, each recognizing a specific epitope, some of which may bepresent on the carrier molecule. Techniques for preparing polyclonalantibodies generally are well known in the art. It is well within theskill of the ordinary practitioner to isolate antibodies which arespecific for the hapten portion of the immunogen. Affinitychromatography is but one method.

Monoclonal antibodies, specific for just one determinant or epitope, maybe prepared eliciting an immune response as before. Followingappropriate incubation and booster injections, B-lymphocyte cells areremoved from the spleens of the animals by standard procedures, and theB-lymphocyte cells are then fused with myeloma fusion partners accordingto standard procedures, such as those described in Kohler and Milstein,"Continuous Culture of Fused Cells Secreting Antibody of PredefinedSpecificity," Nature, 256, 495 (1975).

"Label" as used herein refers to labels capable of providing a directlydetectable signal, as well as to molecules like haptens, which canindirectly be detected. In this way, "label" is interchangeable with"reporter" or "hook". However, at times is necessary to distinguish"label" from a moiety which is capable of generating a measurabledetectable signal, usually an electromagnetic radiation signal. The term"detectable label" or "signaling" label or moiety is used when theintent is to differentiate from hapten-type labels or "hooks".

The term "tracer" refers to a conjugate of hapten with a detectablesignaling label. The tracer permits a determination or assay of theamount of hapten present in an unknown solution. Preferably, the tracersignaling label is a fluorescent molecule as described hereinafter,although the tracer signaling label may encompass other detectablelabels, including by way of example and not limitation, radioisotopes,chemilumiphores and colloidal particles. In an FPIA, the choice of thefluorescent molecule for forming the tracer is advantageously flexibleand is largely up to the preferences of the practitioner. It will bereadily appreciated that the fluorescent labels are ideally chosen inaccordance with their size, that is, the smaller the molecule, the morerapidly it can rotate, and the more effective it is as an FPIA tracercomponent. In the present invention, the preferred fluorescent labelsare fluorescein and fluorescein derivatives. These compounds providefluorescent response when excited by polarized light of an appropriatewavelength and thereby enable the fluorescence polarization measurement.For example, any of the following fluorescein derivatives can be used:fluorescein amine, carboxyfluorescein, a-iodoacetamidofluorescein,4'-aminomethylfluorescein, 4'-N-alkylaminomethylfluorescein,5-aminomethylfluorescein, 2,4-dichloro-1,3,5-triazin-2-yl-aminofluorescein (DTAF),4-chloro-6-methoxy-1,3,5-triazin-2-yl-aminofiuorescein, fluoresceinisothiocyanate. Especially preferred derivatives areaminomethylfluorescein and 5-carboxyfluorescein. Other tracer detectablelabels are also known in the literature, particularly associated withother detection techniques.

The term "oligonucleotide" (sometimes abbreviated to "oligo") refers toshort segments of nucleic acid having a minimum of about 5 nucleotidesand a maximum of several hundred nucleotides. Although, oligonucleotideslonger than about 30 nucleotides are often called polynucleotides, theterm oligonucleotide is used herein to encompass the longer chains aswell. The nucleic acid may be RNA or DNA, although DNA is generallypreferred. The DNA may be natural or synthetic, although the inventionexcels in the automated synthesis of DNA.

Reagents

1 Haptens

a. Structure of Haptens

Haptens which are structurally similar to carbazole or dibenzofuran havethe following general structure: ##STR6## wherein G is O (dibenzofuran)or NR (carbazole), a, a', and A are as defined above.

b. Synthesis of Haptens

Hapten derivatives may be prepared according to routine chemistry usingcarbazole and dibenzofuran core structures commercially available. Theexamples provide further details on synthesis.

Also, it will be recognized by one of normal skill in the an that thereare some limitations for the substituents a and a' when adjacent andtaken together to form a fused ring. The ring fused to the phenyl willnormally have from 4-10 atoms, and preferably from 6-8 atoms.

c. Tethered intermediates

The haptens are converted according to methods known to those skilled inthe art using linker molecules x--L--y, (containing reactive functionalgroups x and y capable of coupling to complementary reactive groups onanother molecule or macromolecule) to produce hapten intermediatecompounds with a tether or side chain, --L--y. Of course, the remainderof the hapten molecule retains a structure substantially similar tothose of the desired determinant(s). Many methods for linking a haptento another molecule are known in the art. Preferred methods involveactivating a functional group on the hapten or linker, and attaching alinker or tether to the hapten via the activated group. The free end ofthe tether, y, is available for coupling to the desired conjugatemolecule, Q. As is known in the art, it is sometimes desirable toactivate a functional group on Q, and sometimes a second linker is used.Depending on the desired y group, it may also be preferred (for ease ofsynthesis) to interchange one y for another (e.g. phosphoramidite orphosphonate for hydroxyl) after the tether has been attached to thehapten.

One possible general activation/coupling scheme is given below. Specificexamples follow for various means for preparing desired products.

The hapten (I) with a hydroxyl group is reacted with an activated linkeror tether molecule x--L--y, wherein x is a leaving group; wherein L isspacer group as defined above; and wherein y is chosen from the groupconsisting of hydroxy, carboxy and amino to form a tethered intermediate(II). For example, the hapten hydroxyl may be reacted in base with ethyl4-bromobutyrate (where x=Br, L=--(CH₂)₃ -- and y=CO₂ C₂ H₅). The tether,--L--y thus becomes --O(CH₂)₃ --CO₂ C₂ H₅). The ethyl ester may bedeprotected to a carboxylic acid by saponification. The carboxylic acidis a reactive group capable of reacting with functional groups in theconjugate partner Q. The reaction conditions for each of these reactionsteps can be obtained from specific examples in the EXAMPLES section orin the literature.

2 Immunogens

a. Structure of Immunogens

Immunogens can be produced from a wide variety of tetheredintermediates. The immunogens of the present invention have thefollowing general structure: ##STR7## wherein a and a', G and A are asdefined above; and Q is an immunogenicity conferring carrier molecule, adetectable label, an oligonucleotide or a solid support.

Typical carriers were previously described. Immunogens find principaluse in the raising of antibodies.

b. Synthesis of Immunogens

In the immunogens of the present invention, the tether functionality, y,of tethered intermediates (II) can be reacted in any of several waysknown to those skilled in the art with the amino groups on a proteincarrier. With carboxyl reactive groups, it is frequently preferable toform amide bonds, which typically are quite stable. Amide bonds areformed by first activating the carboxyl moiety y of the tetheredintermediate by reaction with an activating reagent such as1,3-dicyclohexylcarbodiimide and an additive such asN-hydroxysuccinimide. The activated form of the hapten is then reactedwith a buffered solution containing the immunogenicity conferringcarrier. Alternatively, the carboxylic acid hapten may be converted,with or without isolation, into a highly reactive mixed anhydride, acylhalide, acyl imidazolide, or mixed carbonate and then combined with theimmunogenicity conferring carrier. One of ordinary skill in the art willrecognize that there are many reagents that can be used to form amidebonds other than those listed.

A tethered intermediate with a terminal amine functionality [II, y=--NH₂] can be transformed into a highly reactive N-hydroxysuccinimideurethane by reaction with N,N'-disuccinimidyl carbonate in a suitablesolvent, such as acetonitrile or dimethylformamide. The resultanturethane is then reacted with the immunogenicity conferring carrier in abuffered, aqueous solution to provide an immunogen.

A tethered intermediate with a terminal aidehyde functionality [II,y=--CH(═O)] can be coupled to the immunogenicity conferring carrier in abuffered, aqueous solution and in the presence of sodiumcyanoborohydride, by reductive amination according the methods known tothose skilled in the art.

Alternatively, tethered intermediates containing an alcohol group [II,y=--OH] can be coupled to the immunogenicity conferring carrier by firstreaction it with phosgene or a phosgene equivalent, such as di ortriphosgene or carbonyldiimidazole, resulting in the formation of ahighly reactive chloroformate or imidazoloformate derivative (usuallywithout isolation). The resultant active formate ester is then reactedwith the immunogenicity conferring carrier in a buffered, aqueoussolution to provide an immunogen.

In a manner analogous to immunogens, tethered intermediates can beconjugated to solid supports having functional groups such as amino,hydroxyl or carboxyl groups that are reactive in a complementary sensewith reactive groups, y on the linker of the intermediate. The result isa solid phase which can be used to separate or purify antibodies againstthe hapten.

2 Antibodies

Methods for antibody preparation are generally known and have beensummarized above. Specific examples using the haptens and immunogens ofthe present invention are given in the example section below.

3 Tracers

a. Structure of Tracers

Tracers of the present invention look very much like the immunogensdescribed above, except that Q is a signaling moiety. Tracers have thegeneral structure as described above.

As mentioned above, detectable labels which can be detected inhomogeneous systems are preferred. Particularly preferred arefluorescein and fluorescein derivatives.

Tracers of the invention find use in assays for carbazole anddibenzofuran derivatives, including oligonucleotides derivatized withthis hapten. For tracers of the present invention it is preferred thatthe linker consist of 1 to 12 carbon and heteroatoms. Longer chainsreduce the differential polarization effects by distancing the labelfrom the high molecular weight molecule that modulates its polarizationproperties.

b. Synthesis of Tracers

Tethered intermediates (II) containing an amino group, a carboxyl groupor an alcohol group in the tether can be coupled to fluorescein or afluorescein derivative to prepare the tracers of the present invention.Tethered intermediates with a terminal amine functionality can betransformed into a highly reactive N-hydroxysuccinimide urethane byreaction with N,N'-disuccinimidyl carbonate in a suitable solvent, suchas acetonitrile or dimethylformamide. Or an amine-terminated tetheredintermediate can be activated to an isocyanate. The resultant product isthen reacted with an amino fluorescein derivative to form a urea tracer.An amino-group-containing hapten can also be coupled to acarboxyfluorescein derivative which has been activated withN-hydroxysuccinimide in a suitable solvent.

Tethered intermediates with a terminal carboxyl group on the linker canbe coupled to an amino-terminal fluorescein derivative by firstactivating the carboxylic acid moiety of the tether by reaction with anactivating reagent such as 1,3-dicyclohexylcarbodiimide and an additivesuch as N-hydroxysuccinimide. The activated intermediate is then reactedwith a solution of the fluorescein derivative, resulting in theformation of a tracer. Alternatively, the carboxylic acid hapten may beconverted, with or without isolation, into a highly reactive mixedanhydride, acyl halide, acyl imidazolide, or mixed carbonate and thencombined with the fluorescein derivative.

Alternatively, tethered intermediates containing an alcohol group can becoupled to the fluorescein by first reacting the tethered intermediatewith phosgene or a phosgene equivalent, such as di or triphosgene orcarbonyldiimidazole, resulting in the formation of a highly reactivechloroformate or imidazoloformate derivative (usually withoutisolation). The resultant active formate ester is then reacted with anamino-terminal fluorescein derivative resulting the formation of atracer.

4 Oligonucleotides

a. Structure of Oligonucleotides

Oligonucleotides can be produced from a wide variety of tetheredintermediates.

As noted below, oligonucleotides labeled with haptens find uses innucleic acid hybridization assays, including amplification assays.Haptenated oligonucleotide probes are well adapted for separation and/ordetection of PCR products (see e.g. EP-A-357 011) and/or LCR products(see e.g. EP-A-439 182). In combination with other haptens, (e.g.biotin, fluorescein, dansyl, acetylaminofluorene andiodo-acetylaminofluorene, etc.) probes labeled with the hapten of thisinvention are particularly useful in multiplex versions of PCR and LCR.

b. Synthesis of Tethered Oligonucleotides

In the oligonucleotides of the present invention, the tetherfunctionality y, can be the same as defined above for tracers andimmunogens. Oligonucleotides can be labeled by reacting the yfunctionality with an amino or hydroxyl function of the oligonucleotide,or by direct reaction with the phosphorous via oxidative amination of anH-phosphonate reagent. Amino functionalities are present in the purineand pyrimidine bases, but these sites are less preferred for labelingbecause of their importance in hybridization. Amino functionalities canbe introduced to the 5' and/or 3' ends of oligonucleotides usingreagents such as Aminomodifier® (Clontech, Palo Alto). Hydroxylfunctions are typically formed during automated synthesis.

A preferred method for adding a hapten to the 3' end of anoligonucleotide is disclosed in co-pending, co-owned U.S. patentapplication Ser. No. 630,908, filed Dec. 20, 1990, the disclosure ofwhich has already been incorporated. A preferred method for adding ahapten to the 5' end is through the use of a phosphoramidite reagent asdescribed in Thuong, et al. or Cohen, et al. cited above in theBackground Section.

For example a tethered hapten intermediate (II, y=phosphoramidite) wherethe phosphoramidite, y, is ##STR8## is prepared from the tethered hapten(II, y=--OH] by reaction withN,N-diisopropyl-O-(2-cyanoethyl)chlorophosphoramidite as described inScheme I, below.

The starting compound in Scheme I is an ester of general formula 1wherein W is a spacer group of from 1 to about 50 atoms arranged in astraight or branched chain or cyclic moiety, saturated or unsaturated,with the provisos that (a) not more than two heteroatoms are directlylinked, (b) cyclic moieties contain 6 or fewer members, and (c)branching occurs only on carbon atoms; R¹ and R¹⁰ are independentlyhydrogen, alkyl of from 1-10 carbon atoms, an amino protecting group, oraryl, alternatively R¹ or R¹⁰ when taken together with W and thenitrogen atom to which they are attached may form a cyclic amine.Compound 1 undergoes base hydrolysis in the presence of glyme (step 1),followed by reaction with Meldrum's acid(2,2-dimethyl-1,3-dioxane-4,6-dione, 2) and R² OH (R² is alkyl of from1-6 carbon atoms) to yield the beta-ketoester 3 which is then reduced(step 4) with a borohydride at low temperature, e.g., 15°-30° C. for23-24 hours and neutralized slowly to the diol 4. The neutralizationmust be done very slowly to avoid a dangerously fast release of gas. Theprimary hydroxyl of the diol is then protected by dimethoxytritylation(step 5) to form the tethered hapten 5. The tethered hapten is thenphosphoramidated at the secondary alcohol (step 6) to thephosporamidite-linked hapten 6. The phosphoramidite-linked hapten maythen be used directly to introduce the hapten into a syntheticoligonucleotide at any position. ##STR9##

Alternatively, a tethered hapten intermediate (II, y=phosphonate) wherethe phosphonate is is prepared from the tethered hapten (II, y=--OH] byreaction with phosphorous trichloride followed by hydrolysis. Both thephosphoramidite and phosphonate derivatives are readily incorporatedinto oligonucleotides during synthesis using standard protocols.

Alternatively, a tethered hapten (II, y=--NH₂ ] is reacted in thepresence of carbon tetrachloride with a phosphonate group alreadyincorporated in to the oligonucleotide, via oxidative amidation.

Detailed descriptions of procedures for solid phase synthesis ofoligonucleotides are widely available, e.g., U.S. Pat. Nos. 4,401,796and 4,458,066 which are incorporated by reference. In one embodiment ofthe present invention synthesis of hapten-labeled oligonucleotides isaccomplished by reacting a hapten phosphoramidite with the 5' hydroxylof a nucleotide attached to a growing oligonucleotide chain. The labeledoligonucleotides are purified by standard procedures, e.g., Gait,Oligonucleotide Synthesis: A Practical Approach (IRL Press, Washington,D.C.: 1984).

Of course, it is now fairly routine practice to make oligonucleotides bysynthetic methods in automated synthesizers that are commerciallyavailable, for example Applied Biosystem's DNA Synthesizer 380B.

B. FPIA Assay Methods

The tracers and antibodies raised against immunogens of the presentinvention produce excellent results in a fluorescence polarization assayof the present invention for the semi-quantitative detection of haptenderivatives. The assay is performed in accordance with the generalprocedure of Example 27.

The preferred procedure was designed to be conducted on the TDx®Therapeutic Drug Monitoring System or the ADx™ Abused Drug System, IMx®Fluorescence Polarization and Microparticle Enzyme Immunoassay (MEIA)Analyzer all of which are available from Abbott Laboratories, AbbottPark, Ill. When the TDx, ADx, or IMx systems are used, the assays arefully automated from pretreatment to final reading once the test samplehas been prepared. Manual assays, however, can also be performed.Although the principles of the invention are applicable to manualassays, the automated nature of the TDx, ADx and IMx systems assuresminimal technician time to perform assays and interpret data. Theresults can be quantified in terms of "millipolarization units", "span"(in millipolarization units) and "relative intensity". The measurementof millipolarization units indicates the maximum polarization when amaximum amount of the tracer is bound to the antibody in the absence ofany PCB in the test sample. The higher the net millipolarization units,the better the binding of the tracer to the antibody.

The span is an indication of the difference between the netmillipolarization and the minimum amount of tracer bound to theantibody. A larger span provides for a better numerical analysis of thedata. For the purposes of the present invention, a span of at least 15millipolarization units is preferred.

The intensity is a measure of the strength of the fluorescence signalabove the background fluorescence. Thus, a higher intensity will give amore accurate measurement. The intensity is determined as the sum of thevertically polarized intensity plus twice the horizontally polarizedintensity. The intensity can range from a signal of about three times toabout thirty times the background noise, depending upon theconcentration of the tracer and other assay variables. For the purposesof the present invention, an intensity of about three to about twentytimes that of background noise is preferred, although it is within theskill of the routineer to optimize the signal for each particularsystem.

For fluorescein tracers, the pH at which the method of the presentinvention is practiced must be sufficient to allow the fluoresceinmoiety to exist in its open form. The pH can range from about four tonine, preferably from about six to eight, and most preferably from about7 to 7.5. Various buffers can be used to achieve and maintain the pHduring the assay procedure. Representative buffers include borate,phosphate, carbonate, Tris, barbital and the like. The particular bufferused is not critical to be present invention, but the Tris and phosphatebuffers are preferred.

The preferred FPIA procedure is especially designed to be used inconjunction with the Abbott TDx® Clinical Analyzer, the Abbott TDxFLx™or the Abbott ADx® Drugs of Abuse System, all three of which areavailable from Abbott Laboratories, Abbott Park, Ill. The calibrators,controls, or unknown samples are piperted directly into the sample wellof the TDx® sample cartridge. One of the advantages of this procedure isthat the sample does not require any special preparation. The assayprocedure from this point is fully automated.

If a manual assay is being performed, the sample is mixed with thepretreatment solution in dilution buffer and a background reading istaken. The fluorescence tracer is then mixed with the assay. Theantibody is then finally mixed into the test solution. After incubation,a fluorescence polarization reading is taken.

The fluorescence polarization value of each calibrator, control orsample is determined and is printed on the output tape of an instrument,such as the Abbott TDx® Analyzer, TDxFLx™ or ADx® System. A standardcurve is generated in the instrument by plotting the polarization ofeach calibrator versus its concentration using a nonlinear regressionanalysis. The concentration of each control or sample is read off of thestored calibration curve and printed on the output tape.

With respect to the foregoing preferred procedure, it should be notedthat the tracer, antibody, pretreatment solution, wash solution,calibrators and controls should be stored between about 2 degrees C andabout 8 degrees C while the dilution buffer should be stored at ambienttemperature. A standard curve and controls should be run every twoweeks, with each calibrator and control run in duplicate. All samplescan be run in duplicate.

The preferred reagents, calibrators and controls for a preferredfluorescence polarization immunoassay of the present invention can befound in the Example Section, infra.

C. Methods of Use

Methods of using the novel haptens, tethered intermediates, immunogens,solid supports and tracers have each been described above. Methods ofusing the labeled oligonucleotides include the performance of specifichybridizations, such as sandwich hybridizations known in the art. Forexample, see U.S. Pat. No. 4,486,539 (Ranki) and GB 2 169 403 (Orion).The haptenated oligonucleotides may also be used in amplificationtechniques, such as PCR and LCR. An illustrative use of a haptenatedprimer in PCR is described in EP-A-357 011 (Abbott); the use of ahaptenated probe in LCR is described in EP-A-320 308 and in EP-A-0 439182. Each of the above-mentioned disclosures is incorporated herein byreference.

The invention will now be described by way of examples which areintended to illustrate but not limit the invention.

IV. EXAMPLES

All percentages expressed herein are weight/volume unless otherwiseindicated. Unless stated otherwise, the following abbreviations have themeanings given, and all chemical reagents are from Aldrich Chemical,(Milwaukee, WI).

    ______________________________________                                        ACA         aminocaproic acid                                                 AMF or AMF  aminomethyl fluorescein, a fluorophore                                        (Abbott)                                                          BAE         3 carboxypropyloxy radical Butyric Acid                                       Ester                                                             BSA         Bovine Serum Albumin, an immunogenicity                                       conferring carrier. (Sigma Chemical)                              Celite ®                                                                              A trademark of Manville Products                                              Corporation, for diatomaceous earth                               CDI         1,1' carbonyldiimidazole, a coupling reagent                      DCC         dicyclohexylcarbodimide                                           DEAD or DEADC                                                                             diethylazodicarboxylate                                           DIEA        diisopropylethylamine                                             DMAP        4-(N,N-dimethylamino)pyridine                                     DMEM        Dulbecco's Minimum Essential Medium, a                                        cell culture medium.                                              DMF         N,N-dimethylformamide                                             EBB         ethyl 4-bromobutyrate                                             EEDQ        2-ethoxy-1-ethoxycarbonyl-1,2-                                                dihydroquinoline                                                  glyme       1,2-dimethoxyethane                                               HNQ         8-hydroxy-5-nitroquinoline                                        HOSu or NHS N-hydroxysuccinimide                                              KLH         keyhole limpet hemocyanin, an                                                 immunogenicity conferring carrier                                             (CalBioChem)                                                      NMP         N-methylpyrrolidinone                                             PEG         polyethylene glycol                                               TBAF        tetrabutylammonium fluoride                                       TEA         triethylamine                                                     THF         tetrahydrofuran                                                   ______________________________________                                    

A. Synthesis of intermediates, Tethers, and Tethered Haptens

Example 1: 2-[3-Carboxypropyloxy]-carbazole ethyl ester (G33), atethered hapten.

2-Hydroxycarbazole (5.0 g, 27 mmol), ethyl bromobutyrate (EBB, 5.9 mL,41 mmol), and potassium carbonate (5.7 g, 41 mmol) were refluxed inmethylethyl ketone (50 mL) for 18 h. Tetrahydrofuran (60 mL) was addedto the reaction and it was filtered. The solvent removed under reducedpressure and the solid triturated with hexanes to remove excess EBB. Theyield was 8.1 grams of a light tan solid after drying under reducedpressure. Mass spectrum: DCI/NH₃, (m+H)+@m/z 298, (m+NH₄)+@m/z 315.

Example 2: 2-[3-Carboxypropyloxy]-carbazole (Carbazole-BAE, G40), atethered hapten.

Carbazole ester (1.0 g), prepared above, was dissolved in glyme (30 mL)and aqueous lithium hydroxide (7 mL, 0.5N). After stirring for 24 h atambient temperature, the solvent was removed under reduced temperatureand water (10 mL) was added. The solution was acidified to pH 2.0 withaqueous HC(1N) then extracted with ethyl acetate. The organic extractwas dried and the solvent removed under reduced pressure giving acolored solid (0.82 g). Mass spectrum: DCI/NH₃, (m+H)+@m/z 270,(m+NH₄)+@m/z 287.

Example 3: Active ester of carbazole derivative (G57), a tetheredhapten.

Carbazole-BAE (0.1 g, 0.37 mmol), prepared above (G40), andN-hydroxysuccinimide (0.051 g, 0.45 mmol) were dissolved in NMP (1 mL)then DCC (0.084 g, 0.41 mmol) was added. The reaction was stirred atambient temperature for 18 h then filtered through Celite and diluted to2 mL with NMP. This solution of active ester was used in later examplesbelow.

Example 4: 2-[3-Carboxypropyloxy]-N-[2-thiophenylsulfonyl]-carbazole(CT, H39), a tethered hapten.

Carbazole ester (2.0 g, 6.7 mmol) prepared above (G33) was dissolved intetrahydrofuran (THF, 20 mL) and the solution cooled in an ice bath. Tothe cooled solution, under N₂ atmosphere, was added sodium hydride(0.225 g, 80%, 74 mmol) and the reaction allowed to come to ambienttemperature then heated at 40° C. for 6h. The reaction was cooled in anice bath and a solution of thiophenesulfonyl chloride (1.22 g, 6.7 mmol)in THF (20 mL) was added dropwise over 5 minutes. The reaction wasallowed to come to ambient temperature then heated at 40° C for 18 h,under N₂, protected from light. The solvent was removed under reducedpressure and the residue taken up in glyme (60 mL) and aqueous lithiumhydroxide (15 mL, 0.5N). After 18 h at ambient temperature the solventwas removed under reduced pressure and water (20 mL) was added. Thesolution was acidified to pH 2.0 with aqueous HCl (1N) and the solutionextracted with ethyl acetate. The organic solution was dried with sodiumsulfate and the solvent removed under reduced pressure giving theproduct as a colored solid. Mass spec: DCI/NH₃ ; (m+NH₄)+@m/z 433,(m+NH₄ -H₂ O)+@m/z 415.

Example 5: Active ester of carbazole-thiophene derivative (G58), atethered hapten.

CT (0.2 g, 0.48 mmol), prepared above (H39), and N-hydroxysuccinimide(0.066 g, 0.58 mmol) were dissolved in NMP (1 mL) and then DCC (0.11 g,0.53 mmol) was added. The reaction was stirred under nitrogen atmospherefor 18 h then filtered through Celite. The solution was diluted to 2.5mL with NMP. The active ester solution was used in later examples.

Example 6: 2-[3-t-Butyldimethylsilyloxypropyloxy]-dibenzofuran(Dbf-silyl ether, F141), a tethered hapten.

2-Hydroxydibenzofuran (3.0 gm, 16.3 mmol),t-butyldimethylsilyl-propanediol (3.1 gm, 16.3 mmol), andtriphenylphosphine (5.1 gm, 19.5 mmol) were dissolved in tetrahydrofuran(30 mL). The solution was cooled in an ice bath under N₂ atmosphere andthen diethylazodicarboxylate 3.8 mL, 19.5 mmol) was added slowly. Thereaction was stirred at room temperature for 18 h then the solvent wasremoved under reduced pressure. The residue was purified by columnchromatography (silica gel, 1:9, ethyl acetate/hexanes). Mass spectrum:DCI/NH₃, (m+H)+@m/z 357, (m+NH₄)+@m/z 374.

Example 7: 2-[3-hydroxypropyloxy]-dibenzofuran (Dbf-alcohoi, F145), atethered hapten.

The Dbf-silyl ether (1.0 gm, 2.8 mmol) prepared above (F141) was takenup in dry tetrahydrofuran (2 mL) and TBAF (5.6 mL, 1.0 M in THF, 5.6mmol). The reaction was stirred at ambient temperature for 30 min. thenthe solvent was removed under reduced pressure. The residue wasdissolved in ethyl acetate (40 mL), washed with water (3×12 mL), andsaturated brine (12 mL). The solution was dried over anhydrous. sodiumsulfate and the solvent removed under reduced pressure giving a verypure product. Mass spectrum: DCI/NH₃, (m+H)+@m/z 243, (m+NH₄)+@m/z 260,(m+NH₄.NH₃)+@m/z 277.

Example 8: 2-[3-carboxypropyloxy]-dibenzofuran (Dbf-BAE, F246), atethered hapten.

2-Hydroxydibenzofuran (0.5 gm, 2.7 mmol), ethylbromobutyrate (0.78 mL,5.4 mmol), potassium carbonate (1.1 gm, 8.1 mmol), and sodium iodide (10mg) were combined in 2-butanone (15 mL). The reaction was refluxed untilcomplete, then the reaction filtered and the solvent removed underreduced pressure. The residue was taken up in 6M aqueous KOH/methanol,1:1. After the hydrolysis was complete the reaction was acidified to pH3 and the product extracted with ethyl acetate. The residue was purifiedby column chromatography (silica gel, ethyl acetate/hexanes/acetic acid,10:30:60:0.5). The yield was 0.52 gm of product. Mass spectrum: DCI/NH₃,(m+H)+@m/z 271, (m+NH₄)+@m/z 288, (m+NH₄.NH₃)+@m/z 305.

Example 9: An imidizolide derivative of dibenzofuran (F146a), a tetheredhapten.

Dbf-alcohol (0.275 g, 1.1 mmol), prepared above (F145) was dissolved inNMP (1.5 mL) and then CDI (0.202 g, 1.2 mmol) was added. This providedthe Dbf-alcohol imidizolide (shown below ) which was used as is in laterexamples.

Example 10: 12 Atom Tethered Carbazole Intermediate, a tethered hapten.

N-t-BOC-O-(2-aminoethyl)-2-aminoethanol hydrochloride (0.30 g, 1.25mmol, prepared from 2-(2-aminoethoxy)ethanol by the method of Mattingly,P. G., Synthesis (4):366-68, 1990), Carbazole-BAE (0.335 g, 1.25 mmol),and BOP reagent (0.551 g, 1.25 mmol) were dissolved in acetonitrile (5mL), along with triethylamine (0.521 mL, 3.74 mmol), tetrahydrofuran (3mL), and N-methylpyrrolidinone (2 mL). The reaction was kept at ambienttemperature for 18 h under inert atmosphere. The solvent was removed invacuo and the residue purified by column chromatography using ethylacetate/hexanes as the eluant. Mass spectrum: DCI/NH₃, (m+H)+@m/z 456.The BOC protecting group was removed with trifluoroaceticacid/dichloromethane (5 mL, 1:1) giving the trifluoroacetate salt of theamine.

Example 11: Carbazole Betaketoester, a tethered hapten.

The title compound is prepared according to Scheme 1, steps 1-3, above.The bold-faced compound numbers refer to the compounds in that scheme.

2-Hydroxycarbazole (25 g, 136 mmol), ethyl 4-bromobutyrate (29.3 mL, 205mmol), potassium carbonate (28.3 g, 205 mmol), and sodium iodide (1 g)were refluxed in 2-butanone (250 mL), with vigorous stirring for 48 h.While the reaction was still warm, methylene chloride (750 mL) wasadded. The solution was filtered and the solvent removed in vacuo. Theresidue was washed with hexanes (1 L) with stirring for 30 min thenfiltered. The solid was dried in vacuo giving 38 g of solid. Hydrolysisis done in fresh glyme (35 mL/gram) using aqueous lithium hydroxide(0.5N, 2-4 equivalents). The basic solution is reduced in volume, invacuo, water added, and brought to pH2 with aqueous HCl (1-6N). Theproduct (1) is isolated either by filtration of the solid or byextraction with ethyl acetate.

In a 2 L round-bottom flask, 2-carboxypropyloxy-carbazole (1, 50.0 g,186 mmole) was suspended in dry dichloromethane (700 mL) then Meldrum'sacid (28 g, 195 mmole), diethyl cyanophosphonate (30 mL, 195 mmole) andtriethylamine (52 mL, 371 mmole) were added along with additionaldichloromethane (300 mL). The reaction was stirred for 25 hours atambient temperature under dry nitrogen atmosphere. The solvent wasremoved in vacuo and the residue taken up in ethyl acetate (1300 mL). Tothis solution was added 500 mL of 1.0M phosphoric acid. After vigorousstirring for 30 minutes the precipitate was isolated and washed withwater (7×200 mL). The solid was dissolved in methanol (1.4 L) and thesolution heated to reflux for 5 hours. The solvent was remove in vacuoand the solid triturated in the presence of t-butylmethyl ether. Thesolid was isolated and washed with t-butylmethyl ether (200 mL) thendried in vacuo giving a tan colored solid (48 g, 3).

Example 12: Carbazole-diol, a tethered hapten.

The title compound is prepared according to Scheme 1, step 4 above. Thebold-faced compound numbers refer to the compounds in that scheme.

Carbazole betaketo ester (3, prepared above, 47 g, 144 mmole) wasdissolved in tetrahydrofuran (500 mL) then lithium borohydride (2M intetrahydrofuran, 238 mL, 477 mmole) was added slowly using a cannulawith stirring and under a dry inert atmosphere. After 24 hours, 10%citric acid (500 mL) was added very slowly. The solution was extractedwith ethyl acetate (3×400 mL) and the combined organic extracts washedwith 5% sodium bicarbonate (400 mL) and then concentrated brine (400mL). The solution was dried over anhydrous sodium sulfate, filtered, andthe solvent removed in vacuo giving a white solid (29 g). To purify thediol, it can be taken up in a large amount (2 L) of methanol and thenthe solvent removed in vacuo. This is repeated 4-6 times and then thesolid (4) is dried in vacuo.

Example 13: Carbazole-diol-DMT, a tethered hapten.

The title compound is prepared according to Scheme 1, step 5 above. Thebold-faced compound numbers refer to the compounds in that scheme.

Carbazole-diol (4, prepared above, 7.75 g, 25.9 mmole) was dissolved indry pyridine (70 mL) and then dimethoxytrityl chloride (8.8 g, 25.9mmole), diisopropylethylamine (4.5 mL, 33.7 mmole), andN,N-dimethylaminopyridine (0.16 g, 1.3 mmole) were added. The reactionwas stirred under nitrogen atmosphere, protected from light, for 18 h atambient temperature. The solvent was removed in vacuo and the residuepurified by flash chromatography (6×22 cm bed of silica gel, ethylacetate/hexanes/TEA: 0:100:1, 500 mL; 20:80:1, 1L; 40:60:1, to end).After removing solvent, residual TEA was removed by coevaporation withtoluene and then methylene chloride giving 12.2 grams (>78%) of thetitle compound (5).

Example 14: Carbazole phosphoramidite, a tethered hapten.

The title compound is prepared according to Scheme 1, step 6 above. Thebold-faced compound numbers refer to the compounds in that scheme.

The dry carbazole-diol-DMT (5, 6.0 g, 10 mmole) was dissolved in drymethylene chloride (80 mL) then diisopropylethylamine (7 mL, 40 mmole,distilled from CaH) and 2-cyanoethylN,N-diisopropylchlorophosphoramidite (3.1 mL, 14 mmole) were added. Thereaction was kept at ambient temperature, under nitrogen atmosphere,protected from light for 2 h. Dry methanol (0.6 mL) was added and thenafter 20 min ethyl acetate (200 mL) and TEA (50 mL) were added. Thereaction was extracted with 10% sodium carbonate (2×200mL) and saturatedbrine (2×200mL), dried over sodium sulfate and the solvent removed invacuo. The residue was purified by flash chromatography (3.2×50 cm bedof silica gel, ethyl acetate/hexanes/TEA: 0:100:1, 250 mL to conditioncolumn before applying sample; elution with 0:100:1, 250 mL; 20:80:1,500 mL; 30:70:1, to end). Residual TEA was coevaporated with toluenethen ethyl acetate/methylene chloride, 1:1, giving 6.05 g (76%) of whitesolid (6).

B. Synthesis of Immunogens

Example 15: Carbazole/BSA Immunogen (G59):

BSA (0.5 g) was dissolved in sodium phosphate buffer (pH8.0, 0.05M, 10mL) then NMP (5 mL) was added. Carbazole active ester solution from theexample above (G57)(1.7 mL) was added and the reaction mixed by rotationfor 4 h at ambient temperature. The pH was adjusted with triethylamine(TEA, 0.5 mL) plus aqueous HCl (2 mL, 1.0N). Mixing was continued for atotal of 18 h then the solution was dialyzed against 10% ethanol insodium phosphate buffer (pH8.0, 0.1M, 3×6.6 L), then against distilledwater (3×6L). The solution was lyophilized giving the immunogen as awhite fluffy material (0.51 g)

Example 16: Carbazole-thiophene/BSA Immunogen (G60):

BSA (0.5 g) was dissolved in sodium phosphate buffer (10 mL, pH8.0,0.05M) then NMP (5 mL) was added. To this solution was added CT-BAEactive ester solution (1.6 mL) from example 5 (G58). The reaction wasmixed by rotation, then after 2 h TEA (0.5 mL) was added followed byaqueous HC1 (2 mL, 1N). After 18 h at ambient temperature, the solutionwas dialyzed against 10% ethanol in sodium phosphate buffer (pH8.0,0.1M, 3×6.6 L), and distilled water (3×6 L). The solution waslyophilized, giving a fluffy, white product (0.52 g).

Example 17: Dibenzofuran/BSA Immunogen (G49):

Dbf-BAE (0.085 g, 0.3 mmol) prepared above (F246) and HOSu (0.054 g,0.47 mmol) were dissolved in NMP (2 mL). To this solution was added DCC(0.084 g, 0.41 mmol) and the reaction stirred at ambient temperature for18 h under N₂ atmosphere. BSA (0.5 g) was dissolved in sodium phosphatebuffer (pH8.0, 0.05M) then NMP 2.3 mL) was added. The active estersolution was filtered and added to the protein solution. The reactionwas mixed by rotation for 18 h at ambient temperature. The cloudysolution was dialyzed against sodium phosphate buffer (pH8.0. 0.1M) with10% ethanol (3×6.6 L) at ambient temperature, then with distilled water(3×6 L) at 2°-8° C. Lyopholization gave the immunogen as a white powder(0.47 g).

Example 18: Dibenzofuran/KLH Immunogen (F147):

KLH (0.25 g) was dissolved in phosphate buffer (pH8.0, 10 mL) and thenNMP (4 mL) was added. To this solution was added an aliquot ofimidazolide (0.45 mL, 0.19 mmol), prepared above (F146a). After one houradditional imidizolide (0.30 mL, 0.13 mmol) was added. The reaction wasstirred at ambient temperature for 18 h then dialyzed three timesagainst ethanol/pH8.0 phosphate buffer (1:3, 4 L, 4 times). The solutionwas partially lyophilized then allowed to thaw. The solid was washedwith water and lyopholized giving a grayish solid.

C. Synthesis of Tracers

Example 19: General Tracer Synthetic Procedures

A. General procedure I: A hapten (e.g. 25 mg) is activated withdicyclohexylcarbodiimide (0.07 mmol) and N-hydroxysuccinimide (NHS, 0.2mmol) in tetrahydrofuran (5 mL, freshly distilled from benzophenoneketyl) or dimethyl formamide (5 mL) at 0° C. for 2 h and at ambienttemperature for about 12 h under a nitrogen atmosphere. To an aliquot (1mL) of the activated hapten is added an amino bearing fluoresceinderivative (e.g. 4 mg) along with 2 drops of triethylamine. The reactionmixture is stirred for 12 h, evaporated and chromatographed [e.g.Whatman PLKC18F, 1 mm, 20×20 cm reverse phase plates, methanol/1% aq.acetic acid, 60:40 or MERCK Silica Gel 60 F-254, 2 mm, 20×20 cm,chloroform/methanol, 85:15].

B. General procedure II: A hapten (e.g. 25 mg) is dissolved in thionylchloride (1 mL) and heated to 60° C. for 12 h. Afterwards the excessthionyl chloride is removed in vacuo, leaving the acid chloride of thehapten. The acid chloride is then dissolved in THF (5 mL, freshlydistilled from benzophenone ketyl). To an aliquot (1 mL) of theactivated hapten is added an amino bearing fluorescein derivative (e.g.4 mg) along with 2 drops of triethylamine. The reaction mixture isstirred for 12 h, evaporated and chromatographed [e.g. Whatman PLKC18F,1 mm, 20×20 cm reverse phase plates, methanol/1% aq. acetic acid, 60:40or MERCK Silica Gel 60 F-254, 2 mm, 20×20 cm, chloroform/methanol,85:15].

C. General procedure III: An amino bearing hapten is converted to itshydrochloride by treatment with ethereal hydrogen chloride. The haptenhydrochloride (e.g. 50 mg) is dissolved in THF (10 mL, freshly distilledfrom benzophenone ketyl) and treated with trichloromethyl chloroformate(100 mL) for 30 min under a nitrogen atmosphere. Afterwards thevolatiles are removed in vacuo and the residue is taken up in DMF,divided into aliquots and treated with an amino bearing fluoresceinderivative (e.g. 4 mg) along with one drop of triethylamine. Afterstirring for 12 h, the reaction mixture is evaporated andchromatographed [e.g. Whatman PLKC18F, 1 mm, 20×20 cm reverse phaseplates, methanol/1% aq. acetic acid, 60:40 or MERCK Silica Gel 60 F-254,2 mm, 20×20 cm, chloroform/methanol, 85:15].

Example 20: Carbazole/Aminomethylfluorescein Tracer (G61):

The remainder of the active ester solution prepared above (G57) wasdivided in two and to one part was added a small amount of 5-AMF and adrop of DIEA. After 72 h at ambient temperature, protected from light,the reaction was purified using preparative TLC (reverse phase, C18,1000 μm plates, methanol/500 mM NaCl, 6:4) giving a tracer with thefollowing structure: ##STR10## Mass spectrum: FAB, mH⁺ @613.

Example 21: Carbazole/N-glycyfluoresceinamine Tracer

To a similar portion of active ester solution prepared above (G57) isadded a small amount of N-glycylfluoresceinamine and a drop of DIEA.After about 72 h at ambient temperature, protected from light, thereaction is purified using preparative TLC (reverse phase, C18, 1000 μmplates, methanol/500 mM NaCl, 6:4) giving a tracer with the followingstructure: ##STR11##

Example 22: Carbazole-thiophene/Aminomethyfluorescein Tracer (G62):

The remainder of the active ester solution (G58) was divided in two andto one part was added a small amount of 5-AMF and a drop of DIEA. After72h at ambient temperature, protected from light, the reaction waspurified using preparative TLC (reverse phase, C18, 1000 μm plates,methanol/500 mM NaCl 6:4) giving the tracer shown below: ##STR12## Massspectrum: FAB mH+@759.

Example 23: Carbazole-thiophene/N-glycylfluoresceinamine Tracer:

To a similar portion of the active ester solution prepared above (G58)is added a small amount of N-glycylfluoresceinamine and a drop of DIEA.After about 72 h at ambient temperature, protected from light, thereaction is purified using preparative TLC (reverse phase, C18, 1000 μmplates, methanol/500 mM NaCl, 6:4) giving the tracer shown below:##STR13##

Example 24: Dibenzofuran/Aminomethyfluorescein Tracer (F146b):

An aliquot of the imidizolide (0.6 mL, 0.25 mmol), prepared above(F146a), was mixed with AMF.HCl (0.05 g, 0.13 mmol). After 18 h atambient temperature the residue was purified by preparative TLC (silicagel, 2×2 mm plates, methanol/methylene chloride, 1:9), giving the tracershown below. ##STR14## Mass spectrum: FAB, mH+@m/z 630

Example 25: Dibenzofuran/N-glycyfluoresceinamine Tracer

Dbf-BAE (0.022 mmol), prepared above (F246), and EEDQ (0.024 mmol) aredissolved in NMP (0.3 mL). After 2 h of stirring at ambient temperature,N-glycylfluoresceinamine (0.022 mmol) and DIEA (0.012 mL, 0.067 mmol)are added as in examples 17 and 19 above. After about 18 h at ambienttemperature, protected from light, the solvent is removed under reducedpressure and the residue purified by preparative TLC (reversed phaseC18, 1 mm, methanol/500 mM NaCL, 6:4) giving the tracer shown below.##STR15##

D. Production of Antisera

Example 26

Approximately four to five month old female New Zealand White rabbitswere injected subcutaneously and intramuscularly with an initialinoculation of 0.2 mg of the immunogen in Freund's Complete Adjuvantfollowed by a day 14 boost of 0.1 mg of the immunogen and thereaftermonthly booster injections of 0.05 mg in Freund's Incomplete Adjuvant.Bleeds were taken two weeks following each booster injection and theserum tested for binding to tracers in the TDx instrument. Antibodieswith adequate net millipolarization and span were shown in some bleeds 6weeks from initial inoculation.

E. Production of Hybridomas

Example 27

Four to six week old female BALB/c mice are injected subcutaneously atfour weeks intervals with 0.2 mL of any of the immunogens prepared above(e.g. 5 mg/mL; 0.06 mL of immunogen) in 1.88 mL saline; with 100 mg ofmonophosphoryl lipid A and trehalose dimycloate adjuvant (RibiImmunochem Research, Inc). Three months from initial inoculation, upontesting positive for antibody activity, the donor mice are killed threedays following the last immunization; the spleen is removed asepticallyand placed in a plastic Petri dish with 5 mL of cold Dulbecco's MinimalEssential Medium (DMEM),with 2.0 mM L-glutamine (Medium A ). The spleenis dissociated into a single cell suspension; the cells are centrifugedto a pellet and the red cells lysed by resuspension in 2 mL of 0.83%ammonium chloride in 10 mM Tris buffer. After letting stand for 2 min.,20-30 mL of fresh medium A is added. The cells are washed bycentrifugation and resuspended in 10 mL of fresh medium A.

An immunoglobulin non-secreting mouse myeloma cell line (SP 2/0)deficient in the enzyme hypoxanthine-guanine phosphoribosyl transferase(HGPRT-, EC2.4.2.8), as disclosed by Kearney, Journal of Immunology,1979, 123, 1548, which is incorporated herein by reference, is used asthe fusion partner. The myeloma cell line is maintained in medium A with20% fetal calf serum added. For three days prior to fusion, 0.1 mM8-azaguanine is added to the myeloma cells in order to kill anyHGPRT+revertants. On the day of fusion, the myeloma cells are harvested,washed once in medium A, and resuspended in 5 mL medium A. The myelomaand previously harvested spleen cells are counted using a hemacytometerand their viability assessed by Erythrosin B stain exclusion.

The fusion technique used is modified from that of Gefter el. at.,Somatic Cell Genetics, 1977, 3, 231, which is hereby incorporated byreference. To a sterile 50 mL conical centrifuge tube is added 1-1.5×10⁸spleen cells with an equal number of SP 2/0 myeloma cells. Themyeloma-spleen cell suspension is centrifuged at 1400 rpm for 5 minutesto pellet the cells together. The supernatant is aspirated off and thetube tapped gently to loosen the cell pellet and 1 mL of 50%polyethylene glycol (PEG, MW 1000, Sigma) in DMEM, without serum, isadded to the cell pellet. The cells are resuspended gently in PEGsolution over a period of 1 minute by slowly aspirating up and downusing a 1 mL pipette. The tube is held in the hand for an additional 1minute and then 1 mL of medium A is added slowly to dilute the PEG. Thecells are allowed to stand for an additional 1 minute without agitationor mixing. An additional 20 mL of medium A is added over a period of 3to 5 minutes, and the cells pelleted at 1400 rpm for 5 minutes. Thesupernatant is aspirated off and the cells resuspended in 20 mL ofmedium A with 20% fetal calf serum, 1×10⁻⁴ M hypoxanthine, 4×10⁻⁷ Maminopterin and 3×10⁻⁶ M thymidine (medium C or HAT selective medium).Aminopterin is toxic for cells that lack the enzyme HGPRT and thereforekills all unfused myeloma cells. Fused cells (hybridomas) survive in HATbecause they obtain HGPRT from the B lymphocyte (spleen cell) fusionpartner.

Example 28: Selection of Hybridomas Producing Monoclonal Antibodies

The cell suspension for each of the immunogens prepared above istransferred into a 75 cm2 T-flask and incubated at 37 ° C. in a 5% CO₂incubator for 1-3 hours. The cell suspension is then diluted to 1×10⁶spleen cells/mL with medium C, and 1 mL volumes of the cell suspensionsare added to each well of a 24 well Costar plates. These plates areincubated for 24 hours at 37 ° C. and 5% CO₂. After the incubationperiod 1 mL volumes of feeder cell (non-immunized BALB/c mouse spleencells) suspension in medium C at 2-3×10⁵ cells/mL is added to each ofthe 24 wells of the Costar plates and incubated at 37 ° C, 5% CO₂ for14-17 days. During this period, on alternate days, 1 mL volumes ofmedium is removed from each well by aspiration and replaced with 1 mL offresh medium C. On day 10 the supernatants from the hybridoma containingwells are tested for antibody activity. A few hybridoma suspensions arechosen for further cloning by picking those supernatants whichdemonstrate antibody binding greater than background. For example,binding can be assessed on the TDx analyzer (Abbott Laboratories) usingtracers described in examples 16-21. The cells from wells chosen forcontaining antibody activity are cloned by limiting dilution within 24hours of sampling.

Example 29: Cloning of Hybridoma Culture that Produces MonoclonaiAntibodies.

The cells in antibody secreting wells prepared above are diluted in avolume of medium A and 15% fetal calf serum (medium B) to aconcentration of 10 cells/mL and 100 mL of each diluted cell suspensionare aliquoted into the wells of three Costar plates of 96 wells each.100 mL volumes of feeder cells in medium B at 5×10⁵ cells/mL are addedto each well and the plates incubated at 37° C., 5% CO₂ for 14 days.Supernatants are again tested for antibody activity using an assayprotocol as in the above example. The antibody producing clones are thenexpanded without feeder cells in 24 well Costar plates and finally in 25cm2 T-flasks. 32×10⁶ cells/mL samples of the clone are then stored inmedium B with 10% glycerol added, in liquid nitrogen. 1-2 mL sampleswere then further evaluated for displacement on the TDx instrumentprotocol and one clone is selected for ascites production.

Example 30: In Vivo Production of Monoclonal Antibodies

An in vivo method for obtaining large amounts of monoclonal antibodiesinvolved the adaptation of method described above to grow as an"ascites" tumor. Female BALB/c mice are "primed" by intraperitonealinjection of 0.5 mL of pristane (2,6,10,14-tetra-methylpentadecane).Pristane is a sterile irritant which elicits a serous secretion("ascites") in the peritoneal cavity of mice which acts as a growthmedium. Approximately 4-5 weeks following the pristane injection,aliquots containing 1.5×10⁶ actively growing hybridoma cells harvestedfrom in vitro cultures as described in Example 24 are inoculated intothe peritoneal cavities of primed mice. Seven days following hybridomacell injection, 5-10 mL of ascites fluid is harvested from each mouse.Upon purification by ammonium sulfate precipitation approximately 24.6mg of antibody is obtained per mL of ascites fluid.

Example 31: Fluorescence Polarization Immunoassays

As described previously, the reagents for the FPIA of the presentinvention comprise tracers and antibodies raised against immunogens ofthe present invention, specific for tethered intermediates. In addition,conventionally used assay solutions including a dilution buffer, andhapten derivative calibrators and controls are prepared.

The preferred procedure is designed to be used in conjunction with theautomated TDx, ADx, or IMx systems; however, manual assays can also beperformed. In both procedures, the test sample can be mixed with apretreatment solution and antibody in dilution buffer before abackground reading is taken. The tracer is then added to the testsolution. After incubation, a fluorescence polarization reading istaken.

In the automated assays, the fluorescence polarization value of eachcalibrator, control or test sample is determined and printed on theoutput tape of the TDx, ADx or IMx instrument. The instrument alsogenerates a standard curve by plotting the polarization of eachcalibrator versus it's concentration, using a nonlinear regressionanalysis. The concentration of each control or sample is read off thestored curve and printed on the output tape.

The following reagents are used in the preferred automated haptenderivative assays.

1) the pretreatment solution

2) the tracer diluted in 50% methanol in potassium phosphate buffer(0.15 M phosphate buffer, pH 7.5).

3) the antibody comprising rabbit antisera or mouse monoclonal antibodyraised against a hapten derivative immunogen, diluted in TDx buffer(0.1M phosphate buffer, pH 7.5, containing 0.01% bovine gamma globulinand 0.1% sodium azide) with 30% glycerol;

4) a diluent buffer comprising TDx buffer,

5) a sets of calibrators

6) controls comprising 5 mg/mL hapten derivatives

All polarized fluorescent measurements are made using the TDx instrumentwhich performed the assay in accordance with the following protocol:

1) 22.5 mL of standard or unknown test sample and 12.5 mL each of theantibody reagent and the pretreatment reagent are delivered into thecuvette and a sufficient volume of diluent buffer is added to raise thevolume to 1 mL, and a background intensity reading is taken;

2) 12.5 mL each of pretreatment reagent and antibody, 25 mL of thetracer, and the second 22.5 mL of sample and are added to the cuvette,and a sufficient volume of diluent buffer is added to raise the volumeto 2.0 mL;

3) the reaction mixture is incubated;

4) the fluorescence polarization due to tracer binding to the antibodyis obtained by subtracting the polarized fluorescence intensities of thebackground from the final polarized fluorescence intensities of themixture; and

5) the polarization value for the unknown test sample is compared to astandard curve prepared using calibrators of known hapten derivativecontent.

What is claimed is:
 1. A compound having the following structure:##STR16## wherein a and a' when taken alone are 1 to 4 groupsindependently selected from the group consisting of: hydrogen, C₁ -C₁₀-alkyl, C₁ -C₁₀ -alkoxy, C₁ -C₁₀ -alkylthio, halo-C₁ -C₁₀ -alkyl, C₁-C₁₀ -alkylamino, di-( C₁ -C₁₀ -alkyl)amino, aryl C₁ -C₁₀ -alkyl,optionally substituted aryl, halogen, amino, carboxy, carboxamido,hydroxy, mercapto, nitro, nitroso, sulfo, phospho and protected formsthereof; oralternatively a and a' when adjacent and when taken togetherwith the carbons to which they are joined form a fused ring; G is NRwherein R is hydrogen, C₁ -C₁₀ -alkyl, optionally substituted aryl,optionally substituted sulfonyl, thiophenyl, carboxy, carboxamido, andprotected forms thereof; and A is a linking moiety of the formula--L--y, wherein y is a functional group selected from phophoramidite andphosphonate and protected forms of these functional groups and L isspacer group consisting of from 1 to about 50 atoms.
 2. The compoundaccording to claim 1 wherein a is hydrogen and a' is amino, halogen,hydroxy, nitro, and protected forms thereof.
 3. The compound accordingto claim 1 wherein L is C₁ -C₁₀ -alkyl.
 4. The compound according toclaim 3 wherein a is hydrogen and a' is selected from the groupconsisting of hydrogen, amino, halogen, hydroxy, nitro, and protectedforms thereof.
 5. The compound according to claim 1 wherein G is N and Ris selected from hydrogen, optionally substituted sulfonyl, andthiophenyl.